Gurdon Institute, University of Cambridge, Cambridge, UK.
Nat Protoc. 2012 Oct;7(10):1836-46. doi: 10.1038/nprot.2012.116. Epub 2012 Sep 13.
Efficient derivation of human cerebral neocortical neural stem cells (NSCs) and functional neurons from pluripotent stem cells (PSCs) facilitates functional studies of human cerebral cortex development, disease modeling and drug discovery. Here we provide a detailed protocol for directing the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to all classes of cortical projection neurons. We demonstrate an 80-d, three-stage process that recapitulates cortical development, in which human PSCs (hPSCs) first differentiate to cortical stem and progenitor cells that then generate cortical projection neurons in a stereotypical temporal order before maturing to actively fire action potentials, undergo synaptogenesis and form neural circuits in vitro. Methods to characterize cortical neuron identity and synapse formation are described.
高效地从多能干细胞中分离出人类大脑新皮层神经干细胞（NSCs）和功能性神经元，有助于研究人类大脑皮层的发育、疾病建模和药物发现。本文提供了一种详细的方案，用于指导人类胚胎干细胞（hESCs）和诱导多能干细胞（iPSCs）向所有皮层投射神经元的分化。我们展示了一个 80 天的三阶段过程，该过程可以重现皮层发育，其中人类 PSCs（hPSCs）首先分化为皮层干细胞和祖细胞，然后按照典型的时间顺序产生皮层投射神经元，然后成熟为能够在体外产生动作电位、进行突触形成并形成神经回路的神经元。描述了用于鉴定皮层神经元特性和突触形成的方法。
